Bilirubins are yellow dyes which belong to tetrapyrroles and are present as unconjugated bilirubin and conjugated bilirubins in body fluids. Conjugated bilirubins are those in which sugars (e.g. glucuronic acid) are attached to propionic acid groups of the bilirubins, while the unconjugated bilirubin is bilirubin which is not chemically modified.
In case of a disease like hemolytic anemia or hemolytic jaundice, an amount of the unconjugated bilirubin increases. In case of a disease like obstructive jaundice an amount of conjugated bilirubins increase. Accordingly, the separation and quantitative determination of bilirubins are very important in clinical diagnosis.
There have been proposed many methods for the separation and quantitative determination of bilirubins, such as a method using a diazo reagent, a method using high performance liquid chromatography (HPLC), a method using an oxidizing enzyme such as bilirubin oxidase and the like.
The method using a diazo reagent has many variations due to a sort of a diazotization reaction accelerator and a difference of a quantitative measurement of produced azobilirubins. Typical examples of the methods are a reagent of Malloy & Evelyn et al. (Journal of Biological Chemistry, Volume 119, p 481, 1937) and the like.
The method using high performance liquid chromatography (HPLC) has some variations, such as a Lauff et al. method wherein various bilirubins are eluted and analyzed by the gradient of a phosphate buffer solution and isopropanol, using a reversed phase HPLC column (Journal of Chromatography, Volume 226, p 391, 1981) and the like.
The method using an oxidizing enzyme is a method wherein the bilirubins are oxidized by the oxidizing enzyme to eliminate the absorption of around 450 nm of the bilirubins, whereby an amount of the bilirubins is calculated from the absorption change between before and after the reaction. In this method, the specificity of the oxidation onto the bilirubins can be varied by changing reaction conditions. Reagents for this method include a reagent which contains bilirubin oxidase (Clinical Chemistry, Volume 20, p 783, 1974); a reagent which contains another oxidizing agent, such as laccase, tyrosinase, ascorbic acid oxidase and the like (Japanese Kokai Publication (unexamined) 17999/1984); a reagent for a quantitative determination of all bilirubins, which employs a nonionic surfactant (e.g. cholic acid etc.) or an aromatic carboxylic acid as a reaction accelerator (Japanese Kokai Publication (unexamined) 249060/1984 etc.); and a reagent for a quantitative determination of conjugated bilirubins wherein bilirubin oxidase is only acted on conjugated bilirubins by controlling pH, a buffer solution and a surfactant. Examples of the reagents for conjugated bilirubins are a reagent whereby bilirubin oxidase is used in a buffer solution of pH 9 to 11 (Japanese Kokai Publication (unexamined) 58999/1987), a reagent whereby bilirubin oxidase is used in a buffer solution of pH 5 to 6 which contains an anionic surfactant (Japanese Kokai Publication (unexamined) 152955/1985), a reagent whereby bilirubin oxidase is used in a buffer solution of pH 3.5 to 4.5 (Japanese Kokoku Publication (examined) 44000/1986) and the like. Also, Japanese Kokai Publication (unexamined) 154162/1985 discloses a reagent for the unconjugated bilirubins whereby a ratio of bilirubin in a specimen and bilirubin oxidase is limited within a specified range in a buffer solution of pH 8.0 which contains an anionic surfactant.
In the method using a diazo reagent, all bilirubins and conjugated bilirubins are separately determined by whether a reaction accelerator of the diazotization reaction is contained in the reagent or not. The separation ability, however, is insufficient in practical use and more insufficient for a specimen which does not contain albumin or which contains chemicals, such as salicylic acid. It is also a problem in this method that, since the azobilirubins produced by the diazotization of conjugated bilirubins or the unconjugated bilirubin respectively have different spectrochemical properties, inaccuracy occurs in measured results. Further, ditaurobilirubin which is used as a standard material of conjugated bilirubins is different from conjugated bilirubins in reactivity and spectrochemical properties after diazotization.
The method using HPLC has sufficient selectivity, but requires an expensive and unique apparatus. It also takes a long period of time to measure. The number of specimens to be measured is also limited.
The method using another oxidizing enzyme has a problem in that no adequate standard material is present, the situation of which is similar to the method using a diazo reagent. Since ditaurobilirubin or unconjugated bilirubins are different from conjugated bilirubins in body fluids in spectrochemical properties (e.g. absorptivity coefficient, peak absorption wavelength etc.) or reactivity with a reagent, inaccuracy may occur in measured results. Thus, errors arise when bilirubins in body fluids are separately determined using one standard material. It is also proposed that a combination of ditaurobilirubin and the unconjugated bilirubin is used as a standard material, but a combination ratio has to be selected according to body fluids, and thus its preparation is difficult. The reagents for determining conjugated bilirubins are formulated with the premise that proteins, such as albumin, are present in the specimen. It therefore is difficult for them to precisely measure a specimen which contains a high ratio of bilirubins which are free from proteins, such as serum of a new born baby or serum containing salicylic acid.
Further, Japanese Patent Publication (examined) 44000/1986 discloses a reagent for the quantitative determination of conjugated bilirubin, of which a pH range, however, is disadvantageous for bilirubin oxidase in view of stability and activity.
The method measuring the unconjugated bilirubin, as described in Japanese Kokai Publication (unexamined) 154162/1985, is not suitable for a specimen which contains the unconjugated bilirubin in an unknown concentration, because an amount of bilirubin oxidase should be controlled by an amount of the unconjugated bilirubin. Also, since the reagent used for this method functions not only on the unconjugated bilirubin but on some of conjugated bilirubins, the result of this method is inaccurate.